Concatemers of differentially expressed multiple genes

• US 8,008,459 B2
• Filed: 01/25/2002
• Issued: 08/30/2011
• Est. Priority Date: 01/25/2001
• Status: Expired due to Fees

• Current Assignee: Evolva Holding SA
• Original Assignee: Evolva Holding SA
• Inventors: Goldsmith, Neil, Nielsen, Soren V. S., Sorensen, Alexandra M. P. Santana, Naesby, Michael
• Primary Examiner(s): BURKHART, MICHAEL D
• Application Number:
  US10/466,959
• Publication Number:
  US 20040110174A1
• Time in Patent Office: 3,504 Days
• Field of Search: None
• US Class Current: 536/23.1
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/20 Pseudochromosomes, minichrosomosomes
• CPC Class Codes: C12N 2800/206 of yeast origin, e.g. YAC, 2u

Abstract

In the present invention are disclosed concatemers of concatenated expression cassettes and vectors that enable the synthesis of such concatemers. The concatemer comprises in the 5′→3′ direction a cassette of nucleotide sequence of the general formula [rs2-SP-PR-X-TR-SP-rs1]n wherein rs1 and rs2 together denote a functional restriction site, SP individually denotes a spacer of at least two nucleotide bases, PR denotes a promoter, capable of functioning in a cell, X denotes an expressible nucleotide sequence, TR denotes a terminator, and SP individually denotes a spacer of at least two nucleotide bases, and n>/=2, and wherein at least a first cassette is different from a second cassette. The main purpose of these concatemers is the controllable and co-ordinated expression of large numbers of heterologous genes in a single host. Furthermore, the invention relates to a concatemer of cassettes of nucleotide sequences and a method for preparing the concatemers. In a further aspect, the invention relates to transgenic host cells comprising at least one concatemer according to the invention, as well as to a method for preparing the transgenic host cells. Finally, the invention relates to a vector comprising a cassette of nucleotides, a method for preparing said vector, a nucleotide library comprising at least two primary vectors each comprising a cassette of nucleotides, a method for preparing the library.

Claims

1. A nucleotide concatemer comprising a cassette of nucleotide sequence of the general formula in the 5′

→

3′

direction;

[rs₂-SP-PR-X-TR-SP-rs₁]_n

wherein

rs₁ and rs₂ together comprise a rare restriction site, denoted rs₁-rs₂, comprising a

recognition sequence of 7 to 30 non-N bases,

each SP individually denotes a spacer of at least two nucleotide bases,

PR denotes a promoter, capable of functioning in a cell,

X denotes an expressible nucleotide sequence,

TR denotes a terminator, and

n≧

25, and

wherein at least a first cassette is different from a second cassette.

2. The concatemer according to claim 1, wherein the expressible nucleotide sequence comprises a DNA sequence selected from the group consisting of cDNA and genomic DNA.

3. The concatemer according to claim 1, wherein the rs₁-rs₂ restriction site of at least two cassettes are recognised by the same restriction enzyme.

9. The concatemer according to claim 1, wherein said first cassette differs from said second cassette due to different expressible nucleotide sequences from materials of different expression states.

10. The concatemer according to claim 9, wherein the different expression states represent at least two different species.

11. The concatemer according to claim 9, wherein the different expression states represent at least two different kingdoms.

12. The concatemer according to claim 11, wherein said different expression states represent a eukaryotic species and a prokaryotic species.

13. The concatemer according to claim 1, wherein substantially all cassettes are different.

14. The concatemer of claim 1 wherein rs₁-rs₂ comprises a recognition sequence of at least 8 bases.

15. The concatemer according to claim 1, wherein the expressible nucleotide sequence is obtained through chemical synthesis.

16. The concatemer according to claim 1, wherein the recognition sequence is palindromic.

21. The concatemer according to claim 1, wherein n is at least 30, 75, 100, 200, 500, 750, 1000, 1500, or 2000.

22. The concatemer according to claim 1, wherein n is at least 30 to 60.

4. A cell comprising at least one concatemer of individual oligonucleotide cassettes, each concatemer comprising an oligonucleotide of the following formula in 5′

→

3′

direction;

[rs₂-SP-PR-X-TR-SP-rs₁]_n

wherein

rs₁ and rs₂ together comprise a rare restriction site, denoted rs₁-rs₂, comprising a

recognition sequence of 7 to 30 non-N bases,

each SP individually denotes a spacer of at least two nucleotide bases,

PR denotes a promoter, capable of functioning in the cell,

X denotes an expressible nucleotide sequence,

TR denotes a terminator, and

wherein n≧

25, and

wherein at least two expressible nucleotide sequences are from different expression states.

6. The cell according to any one of claim 4 or 5, wherein substantially all rs₁-rs₂ sequences are recognised by the same restriction enzyme.

7. The cell according to any one of claim 4 or 5, being a yeast cell selected from the group consisting of baker'"'"'s yeast, Kluyveromyces marxianus, K. lactis, Candida utilis, Phaffia rhodozyma, Saccharomyces boulardii, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Candida paraffinica, Schwanniomyces castellii, Pichia stipitis, Candida shehatae, Rhodotorula glutinis, Lipomyces lipofer, Cryptococcos curvatus, Candida spp., Yarrowia lipolytica, Candida guilliermondii, Candida, Rhodotorula spp., Saccharomycopsis spp., Aureobasidiumpullulans, Candida brumptii, Candida hydrocarbofumarica, Torulopsis, Candida tropicalis, Saccharomyces cerevisiae, Rhodotorula rubra, Candida flayeri, Eremothecium ashbyii, Pichia spp., Kluyveromyces, Hansenula, Kloeckera, Pichia, Pachysolen spp, and Torulopsis bombicola.

8. The cell according to claim 7, wherein at least a first cassette is different from a second cassette.

17. The cell according to claim 4, wherein the expressible nucleotide sequence is obtained through chemical synthesis.

18. The cell according to claim 4, wherein the recognition sequence is palindromic.

23. The cell according to any of claim 4 or 5, wherein n is at least 30, 75, 100, 200, 500, 750, 1000, 1500, or 2000.

24. The cell according to any of claim 4 or 5, wherein n is at least 30 to 60.

5. A cell comprising at least one concatemer of individual oligonucleotide cassettes, each concatemer comprising an oligonucleotide of the following formula in 5′

→

3′

direction;

[rs₂-SP-PR-X-TR-SP-rs₁]_n

wherein

rs₁ and rs₂ together comprise a rare restriction site, denoted rs₁-rs₂, comprising a

recognition sequence of 7 to 30 non-N bases,

each SP individually denotes a spacer of at least two nucleotide bases,

PR denotes a promoter, capable of functioning in the cell,

X denotes an expressible nucleotide sequence,

TR denotes a terminator, and

wherein n≧

25, and

wherein rs₁-rs₂ in at least two cassettes is recognised by the same restriction enzyme.

19. The cell according to claim 5, wherein the expressible nucleotide sequence is obtained through chemical synthesis.

20. The cell according to claim 5, wherein the recognition sequence is palindromic.